Fplc machine

Lysogeny broth (LB) medium was purchased from Oxoid Limited (Hampshire, U.K.). Methanol and acetonitrile used for liquid chromatography-mass spectrometry (LC-MS) were high-purity solvents from Concord Technology (Minnesota, U.S.A.). Water used in this work was ultrapure deionized water from Millipore Direct-Q. Chemicals were purchased from Sigma–Aldrich, Geneview, J&K, Amresco or Solarbio. Oligonucleotide primers were synthesized by Tsingke Biological Technology (Beijing, China). DNA plasmid Mini prep, fragment gel extraction and restriction endonucleases clean up kits were from Tiangen (Beijing, China). Talon Cobalt resins were purchased from Clonetech (California, U.S.A.). All protein purification chromatographic experiments were performed on an ‘ÄKTA pure’ or ‘ÄKTA prime plus’ FPLC machine equipped with appropriate columns (GE Healthcare). Protein concentrations were determined according to the method of Bradford [12 (link)] using bovine serum albumin (BSA) as a standard, or using NanoDrop One (Thermo Fisher Scientific). The extinction coefficient for each protein was obtained using the ExPASy ProtParam tool.

SEC-MALS was implemented with an FPLC machine (GE Healthcare, Chicago, IL, USA) connected to a Wyatt MiniDAWN TREOS MALS instrument and a Wyatt Optilab rEX differential refractometer (Wyatt Technology, Santa Barbara, CA, USA). A HiLoad^™ 10/300 Superdex 200 GL (GE Healthcare, Chicago, IL, USA) column was pre-equilibrated with a buffer containing 20 mM Tris-HCl pH 7.5, 150 mM sodium chloride, and 0.5 mM TCEP, and was normalized using ovalbumin. 100 μL of monomer and dimer NDRG3 ΔNC at 2.0 mg/mL were injected into the machine at flow rate of 0.4 mL/min, respectively. Data were analyzed using the Zimm model for fitting static light-scattering data and graphed using EASI graph with a UV peak in the ASTRA V software (Wyatt Technology, Santa Barbara, CA, USA).